The stability of proteins is generally a problem in pharmaceutical industry. It has often been solved by drying the protein in various drying processes, such as freeze-drying. The protein has thereafter been distributed and stored in dried form. The solution before drying or freeze-drying, the dried material and the re-constituted product should all be stable, to avoid a substantial loss of activity in the drying process, as well as during storage or handling. The freeze-drying process is a costly and time consuming process step, which reduces the yield of the product. It would therefore be a great advantage if this step could be avoided when preparing a commercial product. Furthermore, the patient necessarily has to reconstitute the dried protein in a solvent before use, which could be inconvenient for the patient.
Hemophilia is an inherited disease which has been known for centuries but it is only within the last four decades that it has been possible to differentiate between the various forms; hemophilia A and hemophilia B. Hemophilia A is the most frequent form. It affects only males with an incidence of one or two individuals per 10,000 live-born males. The disease is caused by strongly decreased level or absence of biologically active coagulation factor VIII (antihemophilic factor), which is a protein normally present in plasma. The clinical manifestation of hemophilia A is a strong bleeding tendency and before treatment with factor VIII concentrates was introduced, the mean age of the patients concerned was less than 20 years. Concentrates of factor VIII obtained from plasma have been available for about three decades. This has improved the situation for treatment of hemophilia patients considerably and offered them possibility of living a normal life.
A formulation with a low concentration of a protein such as factor VIII, will generally loose activity during purification, sterile manufacturing, in the package and during administration. This problem is usually solved by the addition of human serum albumin (HSA) which reduces the loss of the active protein considerably. HSA functions as a general stabilizer during purification, sterile manufacturing and freeze-drying (see review by Wang et al., J. of Parenteral Sci. and Tech. Vol 42, Number 2S, supplement. 1988). The use of HSA for stabilization of factor VIII is known and is currently used in all highly purified factor VIII products on the market. However, use of HSA is costly and it is desirable to avoid addition of HSA to a therapeutic protein manufactured by recombinant DNA technology. In addition, the use of HSA as a formulation excipient often limits the use of many of the most powerful and sensitive analytical methods for protein characterization.
Several solutions have been proposed for stabilizing various proteins without using HSA. For example, WO-A-94/26286 to Pharmacia proposes reducing the oxygen concentration as a means to improve the stability of factor VIII solutions ready for use. Furthermore, carbohydrates such as disaccharides or sugar alcohols, have been used previously for stabilizing solutions containing conventional factor VIII products with a low purity. Thus, patent specification WO-A-91/10439 to Octapharma discloses injectable solutions containing factor VIII or factor IX, comprising natural or synthetic disaccharides in a concentration of from 0.1 up to 0.9 mol/l.
Carbohydrates such as disaccharides or sugar alcohols, have been used previously also for stabilizing factor VIII compositions during heat treatment for inactivating viruses. Thus, patent specification EP-B-0 117 064 to Green Cross discloses the presence of at least 1,500 mg/ml of a sugar alcohol or a disaccharide stabilizer, preferably sorbitol or saccharose. The process can be carried out for 3 up to 24 hours at 30 to 80.degree. C., or for 1 min at 90.degree. C. The sugar alcohol or disaccharide are removed after the heat treatment, e.g. by ultrafiltration. Patent specification EP-A-0 018 561 to Behringwerke discloses the presence of an amino acid and 20 to 60% (w/w) of a monosaccharide, an oligosaccharide or a sugar alcohol. This corresponds to 500 to 1,500 mg/ml of saccharide or alcohol assuming the specific gravity of the solution to be 1. The process can be carried out for 1 min up to 48 hours, at 30 to 100.degree. C. Patent specification WO-A-94/17834 to Octapharma discloses heat treating compositions containing a protein and a dialkylphosphate or trialkylphosphate for 5 up to 30 hours at 55 up to 67.degree. C. to inactivate viruses devoid of lipid envelopes. The composition can further contain stabilizing agents, such as saccharose, sorbitol or short-chained neutral amino acids.
WO-A-87/00196 to Quadrant Bioresources discloses the use of trehalose for protecting proteins and other macromolecules from denaturation during drying at ambient temperature. The examples reveal that the compounds tested, mainly antibodies and enzymes, are not only stabilized during the drying procedure, but are also stabilized against long-term storage at relatively high ambient temperature. There is no information about either factor VIII or factor IX, nor about aqueous solutions with a reduced concentration of oxygen.
In WO-A-91/18091 to Quadrant Holdings Cambridge it is stated that sugars in general are of limited use for stabilizing biological substances or organic compounds. Specifically, it is stated that non-reducing sugars, such as sucrose, provide very inferior long-term stabilization. To overcome this problem, WO-A-91/18091 discloses the use of certain sugar or sugar derivatives which can be dried without crystallizing and are non-reducing polyhydroxy compounds capable of replicating the effect of trehalose. More particularly, the sugar or sugar derivatives are selected from (i) a non-reducing glycoside of a polyhydroxy compound selected from sugar alcohols and other straight chain polyalcohols, or (i) a non-reducing oligosaccharide selected from raffinose, stachyose and melezitose. There is no information about either factor VIII or factor IX, nor about aqueous solutions with a reduced concentration of oxygen.
It would facilitate the use and manufacture of plasma proteins if the protein could be formulated and distributed to the patient as a stable solution without the addition of albumin and with a prolonged storage life. Also for the patient such a solution would facilitate the handling of the final drug product. The patient could thus administer, e.g. inject, the content of the final drug product directly without reconstitution.